Myosin was modified at the SH1-group with piperidinyl nitroxide, the spin label analogue of iodoacetamide. The proteolytic digestion product of spin-labeled myosin, spin-labeled HMM showed only three-fold actin activation of its Mg-ATPase by actin in contrast to 100-fold activation for unmodified HMM. Furthermore ultracentrifuge experiments demonstrated that when the actin activated ATPase was close to V-max most of the spin-labeld HMM was not bound to actin. These results were also obtained for HMM labeled at the SH-1 with N-ethylmaleimide indicating that the effect of blocking the SH-1 group of myosin does not seem to depend significantly on the nature of the group used for the blocking.